Discovery of an effective anti-inflammatory agent for inhibiting the activation of NF-κB

Abstract In this study, based on the effect of compounds on the activation of NF-κB and NO release, compound 51 was discovered as the best one with NO release inhibition IC50 value was 3.1 ± 1.1 μM and NF-κB activity inhibition IC50 value was 172.2 ± 11.4 nM. Compound 51 could inhibit the activation of NF-κB through suppressing phosphorylation and nuclear translocation of NF-κB, and suppress LPS-induced inflammatory response in RAW264.7 cells, such as the over-expression of TNF-α and IL-6, which were target genes of NF-κB. This compound also showed preferable anti-inflammatory activity in vivo, including alleviating significantly gastric distention and splenomegaly caused by LPS stimulation, reducing the level of oxidative stress induced by LPS, and inhibiting the expression of IL-6 and TNF-α in serum. Thus, it’s reasonable to consider that this compound is a promising small molecule with anti-inflammatory effect for inhibiting the NF-κB signalling pathway.


Introduction
Inflammation is a series of complex defense-related response caused by various injury factors, with red, swelling, heat, pain as the main characteristics 1 . The body's inflammatory response causes cellular changes and immune responses that result in repair of the damaged tissue and cellular proliferation at the injured sites 2 . When inflammation becomes chronic or lasts too long, it may be harmful and lead to disease, even creates an environment that is suitable for the development of various of cancers 3 . Various signalling pathways are key contributors in causing epigenetic changes, and switching on these internal mutations 4 . Therefore, treating the inflammatory causes is always important.
Nuclear factor-jB (NF-jB) is a nuclear transcription factor that plays an important role in cell growth, proliferation, differentiation, apoptosis and carcinogenesis [5][6][7][8] . The NF-jB pathway has long been considered as a typical inflammatory signalling pathway, it regulates proinflammatory cytokine production, leukocyte recruitment, or cell survival, which are important contributors to the inflammatory response 9,10 . It's reported that the activation of NF-jB will induce expression of pro-inflammatory cytokines such as tumour necrosis factor-a (TNF-a), and interleukin-6 (IL-6), which lead to inflammatory diseases, including arthritis 11 , psoriasis 12 , chronic obstructive pulmonary disease (COPD) 13 and inflammatory bowel disease (IBD) 14 . Thus, inhibiting the activation of NF-jB is a promising approach to alleviate inflammation. Currently, several compounds have been identified as NF-jB inhibitors [15][16][17][18] ( Figure 1).
It has been reported that LPS induced the activation of NF-jB, promoting the release of proinflammatory cytokines, such as NO, IL-6 and TNF-a 19 . Excessive NO can induce the development of inflammatory diseases 20,21 . Therefore, inhibiting the excessive secretion of NO effectively is one of the important measures to control the inflammatory response. In this study, we used LPSstimulated RAW264.7 cell model to measure the effect of compounds on NO release, and a dual-luciferase reporter assay to detect the effect of compounds on NF-jB activity in HEK293T cells, which is a cell line with high efficiency of transfection. Through preliminary evaluation of anti-inflammatory activity, we found that compound 6 with the structural characteristics of polysubstituted pyridine in a library of more than 3000 compounds with structural diversity showed good activity and has the potential for modification. Through a series of structural optimisation and biological evaluation, compound 51 (NO release inhibition activity IC 50 ¼3.1 ± 1.1 lM, NF-jB transcriptional inhibition activity IC 50 ¼172.2 ± 11.4 nM) was selected as our title compound. Further studies showed that compound 51 could inhibit LPS-induced phosphorylation of NF-jB p65 and IjB in RAW264.7 cells. In HEK293T cells, compound 51 could block the nuclear translocation of p65 and p50, which induced by TNF-a. Above results indicated that compound 51 could inhibit the activation of NF-jB signal. We also verified the effect of compound 51 on target genes of NF-jB, such as TNF-a and IL-6, the results showed that compound 51 inhibited the expression of TNF-a and IL-6.The results of the in vitro and in vivo experiments showed that the compound is a promising anti-inflammatory compound.

Anti-inflammatory activity and SAR study
Through screening the in-house compound library, compound 6 was found to have effective anti-inflammatory activity, moderate inhibition on LPS-induced NO release, but with undesirable inhibition effect on NF-jB transcriptional activity(NO release inhibition IC 50 ¼19.7 ± 2.6 lM, NF-jB activity inhibition IC 50 ¼1619.7 ± 13.2 nM). Therefore, we identified compound 6 as the HIT compound in this study. In order to obtain more excellent lead compound with anti-inflammatory activity, three rounds of structural modification of compound 6 were successfully implemented.
As listed in Table 1, we first considered to replace the group on the benzene ring at C3 position of pyridine core to observe the change of anti-inflammatory activity. The introduction of methyl (7-9) did not change the activity (NO IC 50 ¼ 15.2-30.2 lM), but the introduction of amino (10-12) significantly improved the inhibitions of NO release (NO IC 50 ¼ 0.9-2.3 lM) and NF-jB reporter activity (NF-jB reporter IC 50 ¼ 95.8-570.9 nM), in which the inhibition of NO release was increased by 10-20 folds, and the inhibition of NF-jB reporter was increased by 3-16 folds. Unfortunately, these three compounds showed strong cytotoxicity, which may not be suitable as lead compounds for the development of anti-inflammatory drugs. Further, the amino groups were replaced with acetylamino groups to obtain compound 13-15. Similar to compounds 10-12, compounds 14 (NO IC 50 ¼ 3.2 lM, NF-jB reporter IC 50 ¼ 167.4 nM) and 15 (NO IC 50 ¼ 1.1 lM, NF-jB reporter IC 50 ¼ 532.5 nM) also showed strong anti-inflammatory activity and strong cytotoxicity. It is gratifying to note that the anti-inflammatory activity of compound 13 (NO IC 50 ¼ 10.2 lM, NF-jB IC 50 ¼ 713.9 nM) was not significantly improved as that of other compounds, but the cytotoxicity is reduced. Next, halogens, trifluoromethoxy, ethyl, methoxy, isopropyl and carbamoyl were introduced to the para position of benzene ring to obtain compounds 16-22. The introduction of halogens (16)(17), trifluoromethoxy (18) and carbamoyl (22) led to the loss of anti-inflammatory activity of the compounds. The introduction of ethyl (19), methoxy (20) and isopropyl (21) did not cause the fluctuation of anti-inflammatory activity, which was consistent with the introduction of methyl (7). Considering the changes in activity and cytotoxicity, compound 13 was identified as the node compound for the next round of transformation, although its activity was not the best.
Next, the 2-aminopyridine core was replaced to give compounds 23-27 to explore the importance of pyridine core. As listed in Table 2, any changes about the 2-aminopyridine led to decreased of inhibitions of NO release and NF-jB reporter activity (Table 2). Therefore, in the third round of transformation, we continued to keep the core of compound 13 unchanged.
Finally, we began to discuss the effect of substituents on the benzene ring at C5 position of pyridine core on the anti-inflammatory activity of compounds. As listed in Table 3, after replacing ethyl with methyl (28, 29) and methoxy (31-34), the anti-inflammatory activity decreased significantly. The introduction of chlorine atom on the para position of benzene ring significantly improved the anti-inflammatory activity of the compound (35 NO IC 50 ¼ 4.2 lM, NF-jB reporter IC 50 ¼ 206.5 nM), but the introduction of chlorine atoms on the meta and ortho positions led to the opposite result. Compounds 38 and 39 showed a similar trend. In addition, compounds 40-48 were obtained by introducing trifluoromethyl, hydroxyl and cyano groups at different positions of the benzene ring. Unfortunately, with the exception of compound 41 (NO IC 50 ¼ 8.4 lM, NF-jB reporter IC 50 ¼ 306.6 nM), none of these compounds exhibited effective anti-inflammatory activity. Finally, in order to explore the possibility of activity change, we obtained compounds 49-52 by introducing poly-substituted benzene rings. Surprisingly, compounds 50 (NO IC 50 ¼ 8.6 lM, NF-jB reporter IC 50 ¼ 577.1 nM) and 51 (NO IC 50 ¼ 3.1 lM, NF-jB reporter IC 50 ¼ 172.7 nM) showed superior anti-inflammatory activity, of which compound 51 was the best. Compared with HIT compound 6, its inhibition of NO release was increased by 6-folds, and the inhibition of NF-jB transcriptional activity was increased by ninefolds. In addition, the compound did not show obvious cytotoxicity. With comprehensive consideration, 51 was identified as the optimal one for further study.

Compound 51 suppressed NF-jB signalling activation
Under normal conditions, inactivated NF-jB is located in the cytoplasm and binds to IjB inhibitors 24 . Activation by inflammatory stimuli leads to the release and nuclear translocation of NF-jB, which shows as the breakdown and phosphorylation of the p65-IjBa complex 25 . As shown in Figure 2(A-C), LPS induced phosphorylation of p65 and IjBa protein, and after pre-treatment with different concentrations of compound 51, the LPS-induced phosphorylation of p65 and IjBa was suppressed significantly in a dose dependent manner. Studies reported that TNFa-activated NF-jB transcriptional activity is accompanied by the nuclear translocation of p65 and p50. Therefore, we tested the effects of compound 51 on TNFa-induced nuclear translocation of p65 and p50 in HEK293T cells by isolating the nuclear fraction. As the result shown, TNF-a induced the appearance of RELA (p65) and NFKB1 (p50) subunits of NF-jB in the nucleus, and this effect of TNF-a was inhibited by compound in a dose dependent manner (Figure 2(D-E)).

Compound 51 suppressed activation of MAPK signalling pathway
MAPKs signalling pathway also plays a key role in the regulation of inflammation 26 . LPS-stimulation activate NF-jB/MAPKs signalling    for different concentrations and then stimulated by TNF-a. The nuclear protein was analysed using WB. ### p < 0.001 compared with control group. ÃÃ p < 0.01, ÃÃÃ p < 0.001 compared to TNF-a group.
pathway, allowing the transcription factors AP-1 translocate into the nucleus and promoting the production of inflammatory mediators 27 . Therefore, we measured the effects of compound 51 on LPS-regulated MAPK signalling activation by WB. As shown in Figure 3(A-D), LPS increased the expression of phosphorylation of P38, JNK and ERK, after treated with compound 51, the phosphorylation level of MAPKs was significantly decreased.
Compound 51 suppressed inflammatory response iNOS is activated in the inflammatory response, which catalyses NO production 28 . COX-2 is also an inflammatory mediator, regulates the activity of NF-jB and other transcription factors, then participates in a series of physiological and pathological processes 29 . We found that the expression of iNOS and COX-2 was  decreased significantly after treated with compound 51 ( Figure  4(A-C)). Activation of NF-jB triggers transcription of various inflammatory factor genes, leading to the release of many inflammation factors such as TNF-a and IL-6, enhancing and amplifying the inflammatory response 30,31 . We detected effect of compound 51 on TNF-a and IL-6. As expected, compound 51 decreased the levels of TNF-a and IL-6 significantly in a dose dependent manner (Figure 4(D-E)). In addition, LPS stimulation will enhance the production of ROS, accumulation of ROS also triggered a series of inflammatory response 32 . We found that compound could decrease the level of ROS obviously (Figure 4(F)), which indicated that compound can alleviate the oxidative stress response induced by LPS. Above results suggested compound 51 could suppress the inflammatory response which induced by NF-jB activation.

Compound 51 alleviated LPS-induced inflammation in vivo
In vitro activity showed that compound 51 significantly inhibited TNF-a induced NF-jB transcriptional activity and LPS-induced inflammatory response. To further evaluate in vivo anti-inflammatory activity of compound 51, we established LPS-induced inflammation model of mice. We found that there were significant changes in organ tissues in mice treated with LPS, including gastric distention and splenomegaly ( Figure 5(A,B)), and these symptoms were relieved after mice were treated with compound. The result of HE staining showed that LPS induced obvious injury of spleen, including broken of nuclear and numerous extramedullary haematopoietic cells. Compound 51 alleviated these injury in a dose-dependent manner ( Figure 5(C)). In addition, after treated with 10 mg/kg LPS, the levels of IL-6 and TNF-a in blood of mice were increased, which suggested there is inflammatory response in mice. While IL-6 and TNF-a levels in mice treated with compound 51 were decreased obviously ( Figure 5(D,E)). SOD and MDA were used as important indicators to evaluate the level of oxidative stress in antioxidant and oxidative capacity, respectively. We found that when compared with control group, SOD activity was decreased and MDA activity was increased in LPS group, compound 51 reversed their activity (Figure 5(F,G)). To confirm compound 51 exert anti-inflammatory effect in vivo through inhibiting NF-jB signalling pathway, we detected the expression of NF-jB in spleen. As shown in Figure 5(H), compound 51 suppressed the activation of NF-jB in splenic organ. The results indicated that compound 51 could alleviate LPS-induced inflammation through inhibiting NF-jB signalling pathway in vivo.

Conclusion
The NF-jB pathway is implicated as a typical inflammatory signalling pathway, its activation leads to a series of inflammatory response. Thus, inhibiting NF-jB activation has long been considered as a promising approach to alleviate many inflammatory diseases. In this study, we synthesised a series of compounds, among them, compound 51 showed favourable NO release inhibition activity (IC 50 ¼3.1 lM) and inhibited NF-jB transcriptional activity strongly. Further studies showed that this compound suppressed LPS and TNF-a induced NF-jB activation, including the effect on phosphorylation of and nuclear translocation of NF-jB. In addition, title compound also exert inhibition effect on MAPKs signal, the expression of phosphorylation of P38, JNK and ERK was significantly decreased. NF-jB activation triggered the downstream inflammatory response, we found that compound 51 decrease the levels of IL-6, TNF-a and ROS, which indicated this compound could alleviate LPS and TNF-a induced inflammatory response through inhibiting NF-jB activation. In vivo study, we evaluated the anti-inflammatory activity of compound 51 using animal model of acute inflammation induced by LPS. LPS stimulation caused changes in organ tissues in vivo, compound reduced inflammatory symptoms, including gastric distention and splenomegaly. The result also showed that title compound decreased the levels of IL-6 and TNF-a in blood. In addition, it was confirmed that this compound could decrease ROS level in vitro, which indicated it could alleviate oxidative stress. So we also measured the activity of SOD and MDA in vivo, two key indicators of evaluate the level of oxidative stress. We found that treatment of compound increased SOD activity and decreased MDA activity. What's more, title compound suppressed the activation of NF-jB signalling in spleen, which indicated that this compound inhibited LPSinduced inflammation through inhibiting NF-jB activity. Overall, we obtained a novel compound with favourable anti-inflammatory activity in vitro and in vivo, which is meaningful for the development of NF-jB inhibitors and anti-inflammatory drugs.

Statistical analysis
The results were expressed as the means ± SDs (SPSS software), and the differences between groups were evaluated using Turkey's method (GraphPad Software). Differences between data were considered significant when the p values was <0.05.

Disclosure statement
No potential conflict of interest was reported by the author(s).